Total Phenolic Content , Total Flavonoid Content , In vitro Antioxidant Activity and Antimicrobial Activity against Human Pathogenic Bacteria of Eremurus Himalaicus – An Edible Herb of North Western Himalayas

Introduction: A number of plants of Kashmir valley are unexplored and Eremurus himalaicus is one amongst them. Eremurus himalaicus is an edible herb of North Western Himalayas which is yet to be evaluated for possession of antioxidant and antimicrobial activities. Method: The antioxidant potential of the plant extracts (Ethyl acetate, EHE; Methanol, EHM and Aqueous, EHA) was assessed by determining the total phenolic content, total flavonoid content, DPPH (1, 1-diphenyl-2-picrylhydrazyl) radical scavenging activity, ferrous metal ion chelating activity, hydrogen peroxide scavenging assay and total reduction capability assay. The plant was further investigated for antimicrobial activity using agar well diffusion method. Results: EHM showed highest total phenolic content followed by EHA and EHE with 270 mg GAE/g, 240 mg GAE/g and 110 mg GAE/g respectively. The flavonoid content was found to be highest in EHA with 85 µg QE/g followed by EHM with 65 µg QE/g and least in ethyl acetate extract with only 20 µg QE/g. Ferrous metal ion chelating potential was also highest for EHA with IC 50 value of 200.336. For DPPH assay and H2O2 scavenging assay the activity was highest for EHM with IC50 value of 148.1788 and 182.3371 respectively. In total reduction capability assay again EHM showed highest reducing power. Besides, antibacterial activity was also screened on some human pathogenic bacterial strains where again the highest activity was shown by EHM. Conclusion: In conclusion, EHM and EHA showed highest antioxidant potential and EHE showed the least. Similarly the antimicrobial potential was highest for EHM showing Eremurus himalaicus as a promising new herb for various human diseases.


INTRODUCTION
A number of human diseases are caused by oxidative stress due to imbalance between the formation and neutralization of pro-oxidants. 1 Oxidation reactions may produce free radicals, which play a vital role in damaging various cellular macromolecules.This damage results into various diseases like arthritis, diabetes, asthma, liver damage, atherosclerosis, inflammation and carcinogenesis. 2Antioxidants terminate these chain reactions by removing free radical intermediates and getting themselves oxidized.Synthetic antioxidants like BHT and Ascorbic acid (Vc) possess good antioxidant potential; however these have some side effects and hence the need for natural antioxidants of plant origin as a potential source of natural antioxidants is the need of the hour.India has a rich biodiversity of plants particularly in the Himalayan belt and many of these plants have not been evaluated for their medicinal properties.One of such plants is Eremurus himalaicus.It is found on the rocky slopes of North-Western Himalayas and its leaves are consumed as food in case of anaemia.It is also used as galactagogue by practitioners of traditional systems of medicines. 3Till date no scientific research work has been done on the medicinal values of this plant and a lot is needed to be explored.So the present work is aimed at assessing the antioxidant and antimicrobial activity of Eremurus himalaicus.

Plant Material Collection and Identification
The first step was the collection of plant from Faqir Goojree area of Dhara, Srinagar, Kashmir at the proper time (in the month of May).
The collected plant material was properly identified and the specimen voucher was deposited in The Centre for Biodiversity and Taxonomy, Department of Botany, University of Kashmir under herbarium number: 1765 (KASH).It was ensured that the plant material was free from soil, pathogens, aflatoxins etc.The material was then dried under shade at room temperature and coarsely powdered in a grinding mill.

Extraction
The coarsely powdered plant material was extracted sequentially with different solvents of increasing polarity (petroleum ether, ethyl acetate, methanol and distilled water) by soxhlet extraction method.The recovered extracts were filtered and the filtrate was concentrated under reduced pressure at 35-45°C using rotary evaporator.The remaining moisture, if present, was removed by keeping the extracts in desiccators and finally stored at 4 o C in well labeled storage vials for further experimental use. 4

Determination of total phenolic content
The determination of total phenolic content was performed using Folin-Ciocalteu method as described by. 5 The positive standard used for the construction of calibration curve was Gallic acid and the range of concentrations taken was from 50 mg/mL to 150 mg/mL.The concentration of phenolics in various extracts was expressed as GAE/g using standard gallic acid calibration curve equation.

Total Phenolic Content, Total Flavonoid Content, In vitro Antioxidant Activity and Antimicrobial Activity against Human Pathogenic Bacteria of Eremurus Himalaicus-An Edible Herb of North Western Himalayas
Determination of total flavonoid content For the determination of total flavonoid content aluminium chloride colorimetric method described by Chang et al., 2002 was employed.The standard used for construction of calibration curve was Quercetin and the range of concentrations that was employed was from 50 mg/mL to 150 mg/mL.The concentration of flavonoids in various extracts was expressed as QE/g using standard Quercetin calibration curve equation.

Determination of Antioxidant Potential
For the determination of antioxidant potential of Eremurus himalaicus various assays were performed on different extracts in triplicates and the mean values were taken.These assays included DPPH assay, H 2 O 2 scavenging assay, Ferrous Metal Ion Chelating Assay and Total Reduction Capability Assay.

Hydrogen peroxide scavenging assay
The hydrogen peroxide scavenging assay was performed according to the methodology of. 7

Ferrous Metal Ion Chelating Activity
The ferrous metal ion chelating potential of various extracts of Eremurus himalaicus was assessed by the method of. 8

Total Reduction Capability assay
The total reduction capability assay was performed using the methodology discussed by. 9

Microbiological Assay
The test organisms that were employed for the microbiological assay are as listed in Table 1.The experiment was performed using Agar Well Diffusion Method according to National Committee for Clinical Laboratory Standards (NCCLS).The experiment was performed in triplicates and mean values were taken.

RESULTS
The total phenolics and total flavonoid content of various extracts of Eremurus himalaicus is shown in Table 3.The results reveal that the total phenolic content was highest in EHM with 270 mg GAE/g followed by EHA and EHE with 240 mg GAE/g and 110 mg GAE/g respectively.The results for the flavonoid content revealed that the highest content of flavonoids are present in EHA with 85 µg QE/g followed by EHM with 65 µg QE/g and least in EHE with only 20 µg QE/g.The antioxidant assays, which were aimed at assessing the potential of extracts of Eremurus himalaicus in prevention of various diseases caused due to oxidative stress, gave variable results.The IC 50 value for DPPH assay was lowest for EHM with a value of 148.18.The respective IC 50 values for other two extracts i.e., EHE and EHA were 235.6 and 161.48 respectively.The results were compared with the reference sample butylated hydroxyanisole (BHA) and ascorbic acid (Vc) which showed an IC 50 value of 81.84 and 48.25 respectively.The IC 50 values in H 2 O 2 scavenging assay were 182.33 (R2:0.995),211.66 (R2:0.995)and 257.33 (R2:0.993)for EHM, EHA and EHE respectively.The IC 50 value for the reference samples Vc and BHA were found to be 81.17(R2:0.96)and 120.21(R2:0.992)respectively.Table 2 shows the ferrous metal ion chelating activity of Eremurus himalaicus.The results reveal that the IC 50 values for EHE, EHM and EHA were 313.28 (R2:0.99),216.87 (R2:0.989)for reference sample EDTA. Figure 1 shows the total reduction capability of EHE, EHM and EHA.As is evident from the figure, there is increase in absorbance with the increase in concentration of the extracts.The antioxidant potential was found to be highest in the reference samples Vc and BHA followed by EHM, EHA and EHE.
The results for the antimicrobial potential of Eremurus himalaicus against human pathogenic bacteria are shown in Table 4.The results reveal that the extract of Eremurus himalaicus with most promising antimicrobial potential is EHE followed by EHM.EHA showed the least antimicrobial potential.The positive reference used was streptomycin (25 mcg) and the negative reference used was DMSO which was also used as a solvent for dissolving the extracts.

DISCUSSION
Total phenolic and flavonoid content, and in vitro antioxidant activity of ethyl acetate, methanol and aqueous extracts of Eremurus himalaicus was determined.Various solvents were used for extraction with increase in their polarity which has been proven to be effective in earlier studies. 10he phytocompounds like flavonoids and phenolics exhibit various biological activities, the most important being the antioxidant activity. 11 Free Radicals and Antioxidants, Vol 7, Issue 1, Jan-Jun, 2017 They directly or indirectly scavenge the free radicals through a series of coupled reactions. 12In our study the extracts of Eremurus himalaicus showed the highest phenolic content in EHM followed by EHA and EHE.However the flavonoid content was highest in EHA followed by EHM.
The flavonoid content in EHE was very less.From this we can conclude that the methanolic and aqueous extracts possess good amount of phenolics and flavonoids which confers antioxidant potential to these extracts.DPPH assay is one of the most widely used methods for screening antioxidant activity.DPPH shows a strong absorption band at 517 nm in visible spectrum (deep violet color).The colour changes to yellow due to the scavenging of DPPH radicals by antioxidant compounds via do-nation of hydrogen atom. 13The absorption decreases and coincides with the number of electrons taken up.The maximum antioxidant potential in this study was observed for EHM in a concentration-dependent manner which was followed by the other two extracts i.e., EHE and EHA respectively.The results were compared with the reference sample BHA and Vc.The scavenging of DPPH radicals can be correlated with the number of available hydroxyl groups.Thus from the results we can infer that the antioxidant activity of EHM may be due to the presence of molecules with an available hydroxyl group.H 2 O 2 is a non-radical molecule that causes oxidative stress to cells.It is produced by dismutation of superoxide anion by univalent reduction.It  In living systems, iron stimulates and accelerates the process of lipid peroxidation. 15However, in presence of chelating agents, there is disruption in the complex formation with the result there is decrease in the color intensity.Thus, the measurement of colour reduction leads to the estimation of the chelating potential or antioxidant potential of the chelating agent.The results for this assay revealed that the extracts of Eremurus himalaicus interfered with the formation of ferrozine-Fe 2+ complex.The ferrous metal ion chelating potential of Eremurus himalaicus followed the order EHE, EHM and EHA.
For the measurement of reductive ability the transformation of Fe3+ to Fe2+ in presence of various extracts of Eremurus himalaicus was examined and it was found to possess considerable reducing ability which serves as an indicator of electron donating potential which represents the antioxidant potential. 15The activity was found to be concentration dependent and the results ascertained that the extracts of Eremurus himalaicus possess significant reducing capacity with EHM having the highest potential.
The second part of the study was antimicrobial activity which was performed by agar well diffusion method.It has been reported that the antimicrobial activity reflects the constituents of the plant extracts. 16lavonoids and phenolics are mostly found to be responsible for the antimicrobial activity of plants. 17,18However, many plants have shown similar antimicrobial activity with different phytocompound combinations.The antimicrobial activity of the Eremurus himalaicus extracts against test organisms revealed that the most promising antimicrobial potential was present in EHE followed by EHM and EHA.The extracts of Eremurus himalaicus showed more inhibitory effect on S. aureus, and P. vulgaris strains of bacteria.However, lesser inhibitory activity was seen for K. pneumonia and P. aeruginosa strains.

CONCLUSION
Eremurus himalaicus, an unexplored plant of Kashmir Himalayas, possesses significant amounts of flavonoids and phenolics, which are important phytocmpounds having various biological activities.Besides, Eremurus himalaicus has good potential of preventing oxidative damage in vitro, establishing the antioxidant activity of this plant.Moreover, the plant also has a capability of preventing the growth of bacteria and hence can be considered a good source for antimicrobial drugs.Overall, Eremurus himalaicus can be considered as a promising new herb that has antioxidant as well as antimicrobial activity and can be a good source for the discovery of new potential drugs.

Figure 1 :
Figure 1: Total reduction capability assay of various extracts of Eremurus himalaicus

Table 4 : Antibacterial activity of various extracts of Eremurus himalaicus using agar disc diffusion method Sample Zone of inhibition diameter (mm)
14ghly important because of its ability to penetrate biological membranes.It causes breaking up of DNA, resulting in single strand breaks and formation of DNA protein cross-links.14Hence,the elimination of H 2 O 2 is very essential for protecting the in-vivo systems.In Hydrogen peroxide scavenging assay the activity is assessed on the basis of decrease in absorbance caused by consumption of H 2 O 2 by antioxidant moieties.Thus we can say that EHM has highest H 2 O 2 scavenging potential followed by EHA and EHE respectively. is